Diseases such as cancer have long been identified by examining tissue biopsies to identify unusual cells. The problem has been that there has been no satisfactory prior-art method to extract the cells of interest from the surrounding tissue. Currently, investigators must attempt to manually extract, or microdissect, cells of interest either by attempting to mechanically isolate them with a manual tool or through a convoluted process of isolating and culturing the cells. Most investigators consider both approaches to be tedious, time-consuming, and inefficient.
A new technique has been developed which can extract single cells or a small cluster of cells from a tissue sample in a matter of seconds. The technique is called laser capture microdissection (LCM). In laser capture microdissection, the operator looks through a microscope at a biological specimen such as a tissue biopsy section mounted on a standard glass histopathology slide, which typically contains a variety of cell types. A capture film is placed over the tissue biopsy section. Upon identifying a group of cells of interest within the tissue section, the operator generates a pulse from a laser. The laser pulse causes localized heating of the thermoplastic film as it passes through it, imparting to it an adhesive property. The cells then stick to the localized adhesive area of the thermoplastic film directly above them. Upon removal of the film from the biopsy tissue, the selected cells or sections of tissue are transferred along with the film. The film can be extracted in order to remove biomolecules for subsequent analysis. Because of the small diameter of the laser beam, extremely small cell clusters or single cells may be microdissected from a tissue section.
By taking only these target cells directly from the tissue sample, scientists can immediately analyze the DNA, RNA, proteins, or other biomolecules in order to characterize the activity of the target cells using other research tools. Such procedures as polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from the tissue sample have been demonstrated.
Laser capture microdissection has successfully extracted cells in many types of tissues. These include kidney glomeruli, in situ breast carcinoma, atypical ductal hyperplasia of the breast, prostatic interepithielial neoplasia, and lymphoid follicles. The direct access to cells provided by laser micro-capture will likely lead to a revolution in the understanding of the molecular basis of cancer and other diseases, helping to lay the groundwork for earlier and more precise disease detection.
Another likely role for the technique is in recording the patterns of gene expression in various cell types, an emerging issue in medical research. For instance, the National Cancer Institute's Cancer Genome Anatomy Project (CGAP) is attempting to define the patterns of gene expression in normal, precancerous, and malignant cells. In projects such as CGAP, laser capture microdissection is a valuable tool for procuring pure cell samples from tissue samples.
The LCM technique is generally described in the published article: Laser Capture Microdissection, Science, Volume 274, Number 5289, Issue 8, pp 998-1001, published in 1996, the entire contents of which are incorporated herein by reference. The purpose of the LCM technique is to provide a simple method for the procurement of selected human cells from a heterogeneous population contained on a typical histopathology biopsy slide.
A typical biological specimen is a tissue biopsy sample consisting of a 5 to 10 micron slice of tissue that is placed on a glass microscope slide using fixation and staining techniques well known in the field of pathology. This tissue slice is a cross section of the body organ that is being studied. The tissue consists of a variety of different types of cells. Often a pathologist desires to remove only a small portion of the tissue for further analysis. Another typical biological specimen is a layer of cells coated from a liquid suspension.
Laser micro-capture employs a transfer film that is placed on top of the tissue sample. The film may contain dyes or pigments chosen to selectively absorb at the frequency of the laser. When the film is exposed to the focused laser beam the exposed region is heated and expands, contacting and adhering to the tissue in that region. The film is then lifted from the tissue and the selected portion of the tissue is removed with the film.
Transfer films such as a 100-micron thick ethylene vinyl acetate (EVA) film available from Electroseal Corporation of Pompton Lakes, N.J. (type E540) have been used. The film is chosen to have a low melting point of about 60° C.-90° C.
While the films employed in laser micro-capture applications have proved satisfactory for the task, a single-layered transfer film has been generally imbued with all of the necessary performance characteristics. For example, the transfer film must be capable of absorbing the optimum amount of energy from the laser for the desired activation of the film. In dye-impregnated films, the optical absorption is a function of its thickness, the type of dye and concentration of dye employed. This property of the film may be in conflict with a desire to select film thickness for other reasons. The film must also expand a desired amount and be capable of adhering to the specimen in desired locations yet substantially avoid adhesion to undesired particles. Furthermore, it is important to keep the temperature of that portion of the transfer film contacting the specimen sufficiently low to avoid damage to or change in the nature of the specimen. Also, the transfer film must be capable of being adhered to a carrier and preferably be transparent to enable observation during all stages of the collection procedures. These performance characteristics, among others, are demanded of the transfer film. The present invention is directed to providing an improved transfer film that de-couples some of the performance characteristics within the transfer film in order to optimize the performance of each.